Human metapneumovirus (HMPV) is a recently discovered respiratory pathogen in the Paramyxoviridae family that infects nearly 100% of the world population. This enveloped virus causes severe viral respiratory disease in infants, the elderly, and immunocompromised patients worldwide. In fact HMPV is estimated to be the second most common cause of pediatric lower respiratory illness, following the closely related respiratory syncytial virus. Furthermore, the rate of hospitalization for respiratory disease caused by HMPV in adults over the age of 65 is near that of influenza. Despite its clinical significance there is no antiviral treatment or vaccine available at this time. Since its discovery several key factors have been identified to play a role in HMPV entry, but much of the mechanisms of binding, fusion, and entry remain unsolved. It has been previously shown by our group that heparan sulfate is a required moiety for HMPV binding and infectivity; however, it is not known if heparan sulfate proteoglycans (HSPGs) serve as a receptor or mediate other necessary cellular factors on the plasma membrane. Furthermore, the route of entry has not been identified for HMPV. Thus, to elucidate the role of HSPGs, we will utilize a collection of currently available HMPV strains from laboratory strains and clinical isolates to determine the effects of heparan sulfate blocking molecules and knockdown studies on specific HSPGs; in order to identify the route of entry, we will utilize viral particles with fluorescent labeled envelopes to track if membrane fusion is occurring at the plasma membrane or within intracellular compartments. Finally we propose to study HMPV entry in the context of traditional cell culture, utilizing physiologically relevant cell lines derived from human lung tissue such as Beas-2B, Calu-3, and A549, as well as novel human airway epithelial tissue, to elucidate important details about its life cycle in this model and identify potential factors that may affect HMPV infection in vivo. We hypothesize heparan sulfate proteoglycans, specifically the syndecan moieties, serve as an attachment factor for HMPV, which is required for internalization, and membrane fusion occurs in intracellular compartments. We expect heparan sulfate is also critical for viral infectivity in human airway epithelium. We also anticipate tissus derived from patients with chronic respiratory disease will show an increased permissibility to HMPV infection.